Method for detecting immune dysfunction in asymptomatic aids patients and for predicting organ transplant rejection

ABSTRACT

A sensitive and accurate tissue culture system and kit fop detecting subtle changes in immune function is provided. The system is based on the comparison of IL-2 production by T helper cells in response to recall antigens including influenza A virus, tatanus toxoid, alloantigens, mouse xenogeneic antigens and the like or combinations thereof. Different stages of immune dysfunction can be differentiated and organ graft rejection can be predicted by the method of the present invention.

This is a division of application Ser. No. 07/535,407 filed Jun. 8, 1990and now U.S. Pat. No. 5,344,755, which is a continuation in part ofapplication Ser. No. 07/341,360 filed Apr. 21, 1990 and now abandoned.

The present invention is related to providing a sensitive and accuratetissue culture system that detects subtle changes in immune functionprior to appearance of symptoms or conditions resulting from immunedysfunction or dysregulation.

It is known that AIDS patients die as a consequence of the failure ofthe immune system. Certain types of cancers, drug-induced immunedeficiencies and disorders related to autoimmunity are some examples ofconditions that result from immune dysfunction. It is vitally importantin such cases to detect and characterize the earliest possible changesin immune function so that timely intervention can take place tocontrol, identify and treat the anomalous condition.

So far, the detection of an immune deficient state in HIV-infectedindividuals who are not yet diagnosed with AIDS has relied mainly on invitro generated T lymphocyte responses to agents such as mitogens andlymphocytes from other donors. These stimuli activate not only the Tlymphocyte population that is primarily affected by HIV (T4 cells), butalso other T lymphocytes (T8 cells), which are not affected by HIV earlyin progression toward AIDS. Studies have been reported using recallantigens, which detect immune deficiency before the development of AIDS(Lane et al. N. Engl. J. Med. 313:79, 1985; Shearer et al. J. Immunol.137:2514, 1986). However, there is no method in these reports todetermine or estimate the time interval between the first detection ofan immune defect and progression to AIDS. Therefore, one does not knowhow early before AIDS diagnosis such defects were detected, nor whetherthese defects represented stages in the progression toward AIDS.Moreover, although these earlier studies measured certain Tlymphocyte-mediated functions, they did not employ a direct measure of Thelper cell function (which is considered to be the primary defect inAIDS patients). Furthermore, the use of recall antigens can givemisleading results, because failure to respond to recall antigens (whichby definition require previous and sometimes recent exposure of thepatient to the antigen) could be due not to HIV-induced immunedeficiency, but rather to the patient not having been previously orrecently exposed to the recall antigen used In the test. Moreover, priorart assays for immune abnormalities in asymptomatic HIV-infectedindividuals do not recognize the possibility of multiple distinctcategories of dysfunction.

In short, the limitations or disadvantages of the prior art tests are asfollows: 1) The time interval for first detection of immune deficiencyand development of AIDS symptoms has not been established: 2) A directmeasure of T helper cell function by assaying for lymphokine productionwas not performed; 3) Recall antigens were used in most studies, whichare dependent on prior and possibly recent antigenic exposure; and 4)Multiple and distinct stages in the loss of T helper cell function thatare predictive for progression toward the disease were neither detectednor characterized.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide a kitfor an in vitro direct test of T helper cell function based onantigen-induced IL-2 production.

It is a further object of the present invention to provide a method fordetecting early signs of dysfunction in individuals afflicted with oneof several different immune system abnormalities.

It is another object of the present invention to provide a method fordetecting early signs of immune dysfunction in individuals asymptomaticof AIDS.

It Is an additional object of the present invention to provide immunefunctional analysis to distinguish at least three distinct stages of Tlymphocyte dysfunction in AIDS.

Other objects and advantages of the present invention will becomeevident from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many of the attendant advantagesof the invention will be better understood upon a reading of thefollowing detailed description when considered in connection with theaccompanying drawings wherein:

FIGS. 1A-1J shows IL-2 production by PBL from two HIV-control donors (A,F), four Walter Reed stage 1 HIV⁺ individuals (B-E), and four WalterReed stage 2 HIV⁺ individuals (G-J). The PBL from these donors wereunstimulated (MED. ∇) or were stimulated with FLU (∘), TET (∘), ALLO(▴), or PHA (Δ). The titration curves represent the dilutions of culturesupernatant used to stimulate the CTLL.

FIGS. 2A-2E shows the mean and standard error values of IL-2 production(detected at a supernatant dilution of 1:4) by PBL from HIV⁻ (top panel)and HIV⁺ (three lower panels) individuals to FLU (▪), TET (), ALLO (□),and PHA (). +/+/+ indicates individuals whose PBL generated normal IL-2responses to all four stimuli: -/+/+ indicates individuals whose PBLwere selectively deficient in IL-2 responses to FLU and TET: -/-/+indicates individuals whose PBL were deficient in IL-2 responses to FLU,TET, and ALLO, but not to PHA; -/-/- indicates individuals whose PBLwere deficient in response to all four stimuli. Number of donors testedis shown in ( ) in donor status column.

FIGS. 3A-3C shows IL-2 production by PBL from three HIV⁺ individualstaken at different time intervals during progressive loss of T_(H) cellfunction. PBL were unstimulated (∇) (mean value of the two unstimulatedcultures), or stimulated with FLU (Δ,▴) or TET (∘,). Open symbolsindicate the response from the earlier bleed: closed symbols indicatefrom the later bleed. The numbers in each panel indicate the two datesthat blood was drawn from a donor to be used in the comparative test.Both samples of the PBL from each donor were cryopreserved and tested inthe same experiment.

FIGS. 4A-4C shows IL-2 production by PBL from a single asymptomatic.HIV⁺ individual (WR 1) taken at three four-month time intervals. PBLwere unstimulated (∇) or stimulated with FLU (), TET ( ), ALLO (▴), orPHA (Δ). The numbers in each panel indicate the dates that the blood wasdrawn and tested. CD4⁺ and CD8⁺ cell numbers are shown in the lower partof each panel.

FIGS. 5A-5E compares IL-2 production by PBL from one HIV⁻ control (A)and four HIV⁺, WR1 patients (B, C, D, E) for response to FLU (∘), TET(), ALLO (□), PHA (▴), and mouse xenoantigens (XENO) (). The titrationcurves represent the dilutions of culture supernatant used to stimulatethe CTLL.

FIG. 6 summarizes the mean values of IL-2 production (detected at asupernatant dilution of 1:4) by PBL from HIV⁻ (upper group of bargraphs) and HIV⁺ (lower four groups of bar graphs) individuals to PHA(□), ALLO (▪), XENO (), TET (), and FLU (). +/+/+ indicates individualswhose PBL generated normal IL-2 responses to all five stimuli; -/+/+indicates individuals whose PBL were selectively deficient in IL-2responses to FLU, TET, and XENO: -/-/+ indicates individuals whose PBLwere deficient in IL-2 responses to FLU, TET, XENO, and ALLO, but not toPHA: -/-/- indicates individuals whose PBL were deficient in response toall five stimuli.

FIG. 7 shows that the FLU response () of PBL from healthy individualsare more sensitive to the in vitro suppressive effects cyclosporin A(CsA) than is the response to ALLO ().

FIG. 8 shows photograph of a 96-well microtiter plate kit that wouldtest leukocytes from eight patients for T helper cell-antigen presentingcell function for unstimulated (medium, MED) and for three T cellstimuli (tetanus toxoid, TET; HLA allogeneic cells, ALLO; and the T cellmitogen, phytohemagglutinin, PHA).

FIG. 9 shows photograph of a modified 96-well microtiter plate kit thatwould test leukocytes from eight patients for T helper cell-antigenpresenting cell function for MED, TET and PHA. Unlike the kit in FIG. 8,lyophylyzed preparations of TET and PHA could be incorporated on theplate prior to distribution.

FIGS. 10A-10F shows the T helper cells tests of asymptomatic, HIV⁺patients before and one month after initiation of AZT therapy. The datafrom three different patients are shown before therapy (left panels) andone month after AZT therapy (right panels). In all three patients theIL-2 response to ALLO was elevated after only one month of AZTtreatment.

DETAILED DESCRIPTION OF THE INVENTION

The above and various other objects and advantages of the presentinvention are achieved by a sensitive in vitro test for T helper cellfunction by measuring IL-2 production to the recall antigens, such asinfluenza A virus, tetanus toxoid, alloantigens and mouse xenogeneicantigens in human peripheral blood leukocytes (PBL) from control andaffected individuals.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described-herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference. Unless mentioned otherwise, thetechniques employed herein are standard methodologies well known to oneof ordinary skill in the art.

In the present study, the T helper cell (T_(H)) function of peripheralblood leukocytes (PBL) from Walter Reed Stage 1 (WR 1) and 2 (WR 2)patients was tested directly by evaluating in vitro production ofinterleukin-2 (IL-2) following stimulation with influenza virus (FLU),tetanus toxoid (TET), mouse xenoantigens (XENO), alloantigens (ALLO),and phytohemagglutinin (PHA). This panel of stimuli was selected forstudy because T_(H) responses to FLU, TET and XENO have been recentlyshown to be MHC self-restricted and require CD4⁺ T_(H) and autologousantigen presenting cells (APC). In contrast, the T_(H) response to ALLOand PHA can utilize both CD4⁺ and CD8⁺ T helper cells. Using thisapproach, three categories of T_(H) dysfunction were identified amongHIV⁺ patients whose clinical stages were WR 1 or WR 2.

GENERAL PROCEDURES AND MATERIALS

Antigen:

Influenza A virus was prepared by infecting chicken eggs with a stock ofinfluenza A virus, and the allantoic fluid was harvested, aliquoted andfrozen at -70° C. Tetanus toxoid was obtained from a commercial source(Massachusetts Department of Health, Boston, Mass.). Mouse lymphocyteswere prepared from the spleens of any strain of mouse for use asxenoantigens. Human peripheral blood leukocytes (PBL) from any healthydonor that is HLA-mismatched with the patient were used as a source ofalloantigens. Optimal stimulatory doses of each antigen were determinedusing PBL from healthy, uninfected donors. Mouse and human cell linesgrown continuously in vitro could also be prepared and used as acommercial resource. Phytohemagglutinin A (PHA) (GIBCO, Grand Island,N.Y.) was used for mitogen stimulation.

Preparation of Lymphocytes for Testing:

PBL from HIV-infected patients and uninfected controls donors wereprepared from whole blood drawn by venopuncture. The PBL were separatedby Lymphocyte Separating Medium (Organon Teknika Corp., Durham, N.C.)and centrifugation at 1800 rpm for 20 minutes, followed by two washingsin RPMI-1640 media (GIBCO, Grand Island, N.Y.). The PBL were eithertested immediately, and/or were cryopreserved (at a freezing rate of 1°C./minute) in liquid nitrogen.

Generation of T Helper Cell Responses:

PBL were diluted in RPMI-1640 medium containing 5% AB⁺ human plasma, andcultured with the above-mentioned stimuli at a concentration of 3×10⁶cells/well in 2 ml. 24-well Linbro plates (Flow Laboratories Inc.,McClean, Va.). The PBL were cultured with the various antigens for oneweek (in the presence of anti-TAC monoclonal antibody, provided by Dr.Thomas Waldmann, NIH, to prevent consumption of IL-2 by the stimulatedPBL). Supernatants were collected at the end of 7 days of culture andwere frozen for later testing for IL-2 content. The supernatants weresubsequently thawed and diluted through five 2-fold dilutions withRPMI-1640 media, and were added to 96-well plates (Flow Laboratories,McClean, Va.) for microcultures of the IL-2 dependent CTLL cell line(American Type Tissue Culture Collection, Rockville, Md.), whichrequires IL-2 for cell proliferation and growth. ³ H-thymidine (ICNInc., Irvine, Calif.) was added to the CTLL cultures 24 hr afteraddition of the supernatants, and incorporation of the isotope wasquantatively determined to measure the IL-2 content of the initialculture supernatants. The concentrations of antigens used forstimulation were: influenza A virus, 1:1000; tetanus, 1:1000; PHA,1:200; HLA allogeneic and mouse xenogeneic stimulating cells wereirradiated with 5000 rad, and added at 2×10⁶ /ml.

It is pointed out that although 3×10⁶ PBL has been used herein per test,the tests could be performed with 2×10⁵ PBL or less which is moredesirable for clinical use.

Patients and Clinical Evaluation

Individuals were diagnosed as being HIV infected if they had anti-HIVantibodies demonstrated by the HIV enzyme immuno-assay (AbbottLaboratories, Irving, Tex.) and confirmed by Western blot analysis(Roche Biomedical Laboratories, Burlington, N.C.). Western blots wereconsidered positive if they showed at least two of the following threebands reactive: p24, gp41, and gp120 or gp160. Patients were classifiedaccording to the Walter Reed Staging System (Redfield et al. The WalterReed staging classification for HTLV-III/LAV infection. N Engl J Med1986: 314:131-2). Walter Reed stage 0 denotes seronegative, high-riskindividuals such as sexual contacts of persons with documented HIVinfection. WR 1 denotes an individual who is seropositive for HIV, buthas more than 400 CD4⁺ T-helper cells/mm³ : WR 2 is similar to WR 1except that the individuals also have presence of chronic adenopathy ofgreater than 1 cm³ at ≧2 extra-inguinal sites; WR 3 identifies patientswith fewer than 400 CD4⁺ T helper cells; WR 4 indicates patients whoadditionally present with impaired delayed skin reactions to a panel ofat least two of five recall antigens; WR 5 denotes the presence ofcomplete anergy or oral candidiasis; and WR 6 is a classification ofopportunistic infection, diagnostic of CDC defined AIDS.

Lymphocyte counts and T cell subsets were determined using laser-basedflow cytometry (Coulter Epics Profile, Coulter Electronics. Inc.,Hialeah, Fla.) and OKT4A (anti-CD4) and OKT8 (anti-CD8) monoclonalantibodies (Orthodiagnostics Systems, Raritan, N.J.).

Skin testing for recall antigens was performed using intradermalinjections of 0.02 ml of PPD (STU) (Connaught Laboratories, Ontario,Canada), Candida albicans (1/500) (Hollister-Stier, Spokane, Wash.),Trichophyton (1/500) (Hollister-Stier, Spokane, Wash.), tetanus toxoid(1/5) (Connaught Laboratories, Ontario, Canada), and mumps (fullstrength) (Connaught Laboratories, Ontario, Canada).

EXAMPLE 1 In Vitro Tests for T_(H) Function

Whole blood from HIV⁻ and HIV⁺ individuals was drawn in Vacutainer tubescontaining preservative-free heparin (Becton-Dickinson, Rutherford,N.J.) and shipped from Lackland, Tex. to Bethesda, Md. overnight atambient temperature (22°-24° C.) in crushproof containers. PBL wereseparated on lymphocyte separation medium (LSM: Organon Teknika Corp.,Durham, N.C.). The separated PBL were washed twice in phosphate-bufferedsaline, and the number of viable cells was determined by trypan blueexclusion and hemacytometer. Cells were then resuspended at 3×10⁶ /ml inRPMI 1640 (GIBCO, Grand Island, N.Y.) containing 0.5 percent penicillinand 1 percent glutamine. One ml of PBL was added per well to 24-wellflat-bottom Linbro tissue culture plates (Flow Laboratories, Inc.,McClean, Va.). The PBL were cultured without stimulation or werestimulated with: a) influenza A/Bangkok RX73 (at a final dilution of1:1000) as described by Shearer et al. J Immunol 1986, 137:2514-21; b)tetanus toxoid (at a final dilution of 40 lf/ml) (MassachusettsDepartment of Health, Boston, Mass.); c) irradiated (5000 rad) mousespleen cells (2×10⁶ /well): d) a pool of irradiated (5000 rad) PBL fromtwo or more unrelated HIV⁻ donors (2×10⁶ /well for IL-2 production and2×10⁵ /well for proliferation); and e) PHA (GIBCO) diluted 1:200. PooledAB⁺ plasma was added to each well (final dilution 1:20). Supernatants ofstimulated and unstimulated cultures were harvested 7 days later andfrozen at -20° C. For studies of IL-2 production, the anti-IL-2 receptorantibody, monoclonal anti-TAC (obtained from Dr. T. A. Waldmann,Metabolism Branch, NCI, NIH, Bethesda, Md.) was added at the initiationof culture at a final concentration of 10 μg/ml, in order to block IL-2consumption (Uchiyama et al, J Immunol 1981, 126:1393-7). Thesupernatant IL-2 activity was assessed as the ability to stimulate theproliferation of the IL-2-dependent cell line, CTLL. This cell line isstimulated by human IL-2, but not by human IL-4. Assay culturesconsisted of 8×10³ CTLL/well and five successive 2-fold dilutions ofsupernatant. Twenty-four hours later, the cultures were pulsed with 1 Ciof ³ H!thymidine (ICN Radiochemicals, Irvine, Calif.) and harvested 18hours later. Results are expressed as mean cpm for three replicate wellsfor a given supernatant dilution. Standard errors were always less than10 percent of the mean values. The concentration of anti-Tac antibodyused in the initial culture did not inhibit CTLL proliferation.

Determination of Responsive and Unresponsive Patients

Patients were defined as responsive to a given antigen if the mean cpmof their stimulated cultures was greater than three standard deviationsabove the mean unstimulated cpm of the HIV⁻ control donors. The cutoffvalue for IL-2 production was 7300 cpm and was derived from a mean of 70HIV⁻ donors. Supernatant dilutions of both 1:2 and 1:4 were used fordetermination of responsiveness for the Il-2 assay.

Statistical Analysis of Data

Row (R) x column (C) contingency tables were set up as shown below inTables 2-4 for testing possible correlations of the four different T_(H)functional categories with CD4⁺ cell numbers or WR staging (Snedecor etal, Statistical Methods, 7th Edition. Ames, IA: The University of IowaPress, 1980). The sum of the χ² value was calculated by the sums of theequation χ² =(f-F)² /F, where f is the observed frequency and F is theexpected frequency. The degrees of freedom for this analysis are:df=(R-1)(C-1). Student t tests were performed for the comparisons of twoindependent samples of unequal size as described by Snedecor and Cochran(Snedecor et al. supra), and p values were determined.

RESULTS Patterns of T_(H) Responses in WR 1 and WR 2 Patients

Peripheral blood leukocytes from 70 HIV⁻ control donors and 74 HIV⁺ WR 1and 2 patients were tested for in vitro production of IL-2 followingstimulation with FLU, TET, ALLO, or PHA. The complete IL-2 titrationcurves for each of these stimuli are presented in FIG. 1 for two HIV⁻controls (FIG. 1, panels A and F), four WR 1 patients (FIG. 1, panelsB-E), and four WR 2 patients (FIG. 1, panels G-J). The data on thesepatients were selected from the group of 74 to illustrate four differentpatterns of IL-2 responsiveness. The first pattern, observed in both WR1 and WR 2 patients (FIG. 1, panels B and G), is characterized bypositive IL-2 responses to all four stimuli. However, in many of thepatients, the responses to FLU and TET were moderately below thoseobtained using PBL from the HIV⁻ controls (compare FIG. 1, panels B andG with A and F, respectively). In the second pattern. PBL from WR1 andWR2 patients (FIG. 1, panels C and H) generated near normal IL-2responses to ALLO and PHA, but failed to produce IL-2 in response to FLUand TET. In the third pattern, PBL from the WR1 and WR2 patients shownin FIG. 1, panels D and I, responded to PHA but failed to respond toFLU, TET, and ALLO. Finally, PBL from the WR1 and WR2 patients shown inFIG. 1, panels E and J, failed to respond to any of these stimuli. Theseresults indicate that four different states of T, function can beidentified within WR 1 and WR 2 patients, and that Walter Reed criteriado not predict the in vitro T_(H) functional potential of thesepatients. Furthermore, the failure of WR 1 and WR 2 patients to respondto any combination of these stimuli did not appear to be correlated withCD4⁺ cell numbers (see below).

Using the criteria described herein supra, the number of individuals inthe group of 74 patients and 70 controls were determined who respondedto FLU, TET, ALLO, or PHA by IL-2 production. Forty-nine of these samepatients and 58 of the controls were also tested for proliferativeresponses to the same stimuli. The numbers and percentages of patientsand controls who were unresponsive to each of the stimuli by either ofthe two T_(H) tests are presented in Table 1. Sixty-two of the 74 HIV⁺donors (84 percent) failed to respond to FLU or TET by the IL-2 test.There was an exact correlation between the IL-2 responses to FLU and TETIn that the same 62 patients who failed to respond to FLU also did notrespond to TET. A much lower proportion of these donors was unresponsiveto ALLO (30 percent), and an even lower proportion was unresponsive toPHA (14 percent). In contrast, only one HIV⁻ control donor wasunresponsive to FLU and TET, and none of the controls was unresponsiveto ALLO or PHA. The percent of HIV⁻ controls that was unresponsive toFLU and TET was only 1 percent. All of the controls responded to ALLOand PHA by both assays. For convenience, those patients who responded toall four stimuli were referred to as +/+/+ (FLU and TET/ALLO/PHA); thosewho failed to respond to FLU and TET, but responded to ALLO and PHA as-/+/+; those who failed to respond to FLU, TET, and ALLO, but respondedto PHA as -/-/+; and those who did not respond to any of the stimuli as-/-/-. It should be noted that no patients were found who: a) respondedto FLU or TET but not to ALLO or PHA: or b) did not respond to PHA butwere responsive to any of the other three stimuli. These results arecompatible with a pattern of sequential progression from +/+/+ to -/+/+,to -/-/+, and finally to -/-/-.

FIG. 2 lists the frequencies of these four categories of T_(H)responsiveness, and within each category, quantitatively summarizes theIL-2 results obtained from each stimulant. The data shown are for asupernatant dilution of 1:4; similar results were obtained at dilutionsof 1:2 and 1:8. Despite the fact that 12 HIV⁺ patients tested positivefor IL-2 production in response to each of the four stimuli (+/+/+),their mean cpm values for FLU and TET stimulation were 2-fold belowthose of the HIV⁻ controls (compare FIG. 2, panels A and B). Among the40 patients who were selectively unresponsive to FLU and TET (-/+/+),IL-2 production levels to FLU and TET were significantly below those ofthe HIV⁻ controls (compare FIG. 2, panels C and A; p<0.01 for TET), andalso significantly below those of the responsive HIV⁺ patients (compareFIG. 2, panels C and B: p<0.05 for FLU and p<0.05 for TET). Responsesdid not differ significantly among these three groups for responses toALLO and PHA (FIG. 2, panels A-C), The 12 patients who responded only toPHA by IL-2 production (-/-/+ exhibited a significant reduction in meancpm for ALLO when compared with the ALLO response of -/+/+ patients orthe HIV⁻ donors (compare FIG. 2, panel D with C and A; p<0.05). The meancpm for response to PHA, however, was equivalent in these two groups.Also, there was a further decline in the magnitude of responses to FLUand TET in -/-/+ patients. Finally, the 10 patients who wereunresponsive to any of these four stimuli (-/-/-) exhibited asignificant reduction in cpm for IL-2 production to PHA compared to thegroups of patients who responded to PHA (compare FIG. 2, panel E with A,B, C, and D; p<0.05). There was a further reduction in cpm for responseto ALLO in this totally unresponsive group (compare FIG. 2, panel E withD). The results of FIG. 2 verify that these 74 WR 1 and WR 2 patientscan be divided into four functional categories, based on T_(H) responsesto recall antigens, alloantigens, and a T-cell mitogen.

Lack of Correlation Between T_(H) Function and Either CD4⁺ Cell Numberor WR 1 and WR 2

To determine whether there was a correlation between any of the fourT_(H) functional patterns and CD4⁺ cell numbers of either of the earlyWalter Reed stages, contingency tables were prepared in which thenumbers of patients in each functional category are shown for threeranges of CD4⁺ cell numbers (Table 1) and for WR 1 and WR 2 patients(Table 2). Patients with CD⁴⁺ cell numbers below 400/mm³ are notincluded, because such patients are, by definition, in WR 3 or higher.Statistical analysis of the data in the R×C contingency comparison ofTable 2 indicate that there was no correlation between any of the fourT_(H) response patterns (by IL-2 or proliferation) and the CD4⁺ cellnumbers. A similar analysis of the frequency data for the four T_(H)categories by either the IL-2 production or proliferation assays, forT_(H) function indicates no correlation of function with WR1 or WR2(Table 2).

Time-Dependent Changes in T_(H) Function

The data illustrated in FIGS. 1 and 2 suggest a progression from thefunctional stage in which the patient is responsive to all four stimuli(+/+/+), through a selective loss in T_(H) function to recall antigenssuch as FLU and TET (-/++), followed by loss of response to ALLO(-/-/+), and finally to a totally unresponsive state (-/-/-). However,these data, as well as the data collected on most of the 74 patients,represent only one point in time for each individual's progressiontoward symptomatic AIDS.

A longitudinal comparison of FLU-stimulated IL-2 production by PBL fromthree donors who have been involved in this study for several years isshown in FIG. 3. PBL from the donor shown in panel A were cryopreservedin 1983 and in 1988. This donor remained asymptomatic throughout the4.5-year period and had 573/mm³ of CD4⁻ cells at the time of the lastblood collection. PBL from the donor in panel B were cryopreserved in1984 and in 1987. This donor experienced a reduction in CD⁴⁺ cells (from500 to 139/mm³) during this period, but was not diagnosed withlymphadenopathy or AIDS. PBL from the donor in panel C werecryopreserved in 1985 and 1988. During this time interval, this donorexperienced a reduction in CD4⁻ cells from 715 to 235/mm³, but remainedotherwise asymptomatic. The two preparations of PBL from each of thesethree donors were thawed simultaneously and tested for their ability togenerate IL-2 in response to stimulation with FLU and TET (donor inpanel A) or with FLU only (donors in panels B and C). The results (FIG.3) illustrate that all three donors had lost the ability to generate aT_(H) response to FLU during the 3- to 4.5-year period in which theseblood samples were drawn. These results demonstrate that in these threedonors there was a time-dependent, selective loss of T_(H) function toCD4-dependent antigens such as FLU and TET and suggest that a similarpattern of selective loss of T_(H) response occurred in the other donorswho were selectively defective in their responses to FLU and TET. Itshould be noted that the time-dependent loss of T_(H) response to FLUand TET in the donor in panel A was not due to a critically low numberof CD4⁺ cells. Due to limitations on the number of cryopreserved PBLavailable, it was not possible to test these individuals for T_(H)function to ALLO or PHA.

To obtain a more precise time estimate for development of T_(H)unresponsiveness, an asymptomatic, HIV⁺ donor was studied at four-monthintervals to detect possible loss of T_(H) function. FIG. 4 illustratesthe titration curves for IL-2 production by PBL stimulated with FLU,TET, ALLO, or PHA. In March 1988, the patient was marginally responsiveto FLU and TET, but was strongly responsive to ALLO (FIG. 4, panel A).(PHA was not tested in this experiment.) During the next 8 months, thepatient's FLU and TET responses continued to decline to unstimulatedlevels whereas the ALLO and PHA responses remained strong (panels B andC). During this time interval, the patient did not develop any symptomsand CD4⁺ and CD8⁺ T cell numbers increased slightly. In all threeexperiments. PBL from HIV⁻ control donors generated potent T_(H)responses to all four stimuli (data not shown). These data demonstratethat loss of T_(H) function to recall antigens can occur abruptly andare not necessarily associated with symptomatic changes nor with areduction in CD4⁺ cell numbers.

To further investigate possible progression in T_(H) defects, IL-2responses of PBL from patients in the more advanced WR 3 to WR 6 werestudied. If the failure to produce IL-2 in response to ALLO and PHArepresents T_(H) dysfunction associated with more advanced stages ofAIDS progression, then it would be expected that a larger proportion ofWR 3 to WR 6 patients would be unresponsive not only to FLU or TET, butalso to ALLO or PHA. The proportions of IL-2 responses by 22 patientsare summarized in Table 3 and are compared with the proportions ofresponses by our 74 WR 1 and WR 2 patients using the same two stimuli.Whereas 16 percent of the WR 1 and WR 2 patients responded to FLU, 0percent of the WR 3 to WR 6 patients responded to FLU. Furthermore,fewer WR 3 to WR 6 patients responded to ALLO than did WR 1 and WR 2patients. In fact, the majority of the WR 3 to WR 6 failed to respond toFLU or ALLO (64 percent) compared with only 30 percent of the WR 1 andWR 2 patients who failed to respond to both stimuli. The statisticallikelihood that these frequency differences between WR 1 and WR 2 and WR3 to WR 6 patients were due to chance was less than 1 percent. Thus,these results demonstrate that the T_(H) functional phases shown inFIGS. 1 and 2 represent a sequential progression of immune dysfunctionthat is associated with progression toward AIDS.

It should be pointed out that earlier studies have reported that theT_(H) and CTL responses to recall antigens may be defective in certainHIV⁺ patients. For example, Lane et al (N Engl J Med 1985, 313:79-84),using enriched CD4⁺ T cells from one AIDS patient, demonstrated aqualitative defect in the proliferative response to TET but not to PHA.Smolen et al (J. Clin Invest 1985; 75:1818-34) and Garbrecht et al (ClinExp Immunol 1987; 67:245-51), respectively, demonstrated defects in IL-2production and proliferation in patients for the autologous mixedlymphocyte reaction but not to HLA alloantigens. Shearer et al (JImmunol 1986; 137:2514-21) have previously demonstrated a selectivedefect in CTL responses to FLU but not to ALLO in asymptomatic, HIV⁺individuals. Because the deficient CTL response to FLU could becorrected in vitro by recombinant IL-2, the CTL defect appeared toreflect an underlying deficiency in CD4⁺ T_(H) function. The presentstudy measures T_(H) function directly by assessing IL-2 production andextends these findings by: (a) identifying a series of three distinctT_(H) functional defects in asymptomatic patients: (b) demonstrating atime-dependent progressive and selective loss of T_(H) function inindividual asymptomatic patients; and (c) attempting to correlate thesedefects with clinical staging criteria.

The three different patterns of T_(H) dysfunction identified among theseasymptomatic patients were (a) a selective loss of T_(H) function torecall antigens such as FLU and TET, but retention of T_(H) functionupon stimulation to ALLO or PHA (54 percent); (b) an absence of T_(H)function to the recall antigens and to ALLO, but preserved responses toPHA (16 percent); and (c) a lack of response to all of the stimuli (14percent). Not all asymptomatic, HIV⁺ individuals exhibited thesedefects, because 16 percent of these asymptomatic patients wereresponsive to all of the stimuli, None of the three patterns of T_(H)dysfunction could be attributed to a critical reduction in CD4⁺ cellnumbers. In fact, a large proportion of these patients had CD4⁺ cellfrequencies in excess of 600/mm³ (58 percent). It is estimated that thetime interval between the onset of the T_(H) functional defect to recallantigens and identification of AIDS symptoms can be as long as twoyears, However, the data presented in FIG. 4 (from an asymptomatic WR 1patient) demonstrate that the selective loss in T4 T helper cellfunction to recall antigens such as FLU and TET can occur within therelatively short period of eight months and is not associated with areduction in T4 cell numbers, a parameter commonly used in followingHIV-infected patients.

The T helper cell responses to FLU and TET require pre-immunization(i.e. they are recall antigens), whereas the response to allo does not.Thus, failure to respond to FLU and/or TET could reflect lack of recentexposure to these recall antigens rather than necessarily being due toan induced immune defect. To avoid this potential flaw in the detectionsystem, T helper cell reponse to mouse xenoantigens (XENO) has beenadded to the battery of antigens that are to be used, because it hasbeen found that the response to XENO (which is not a recall antigen)utilizes the same T4 T helper cell pathway used by the recall antigensFLU and TET, but does not use the alternate helper pathways that can beemployed by the ALLO response. Therefore, it would be expected that theT helper cell responses of PBL from HIV⁺ individuals would follow thepatterns of FLU and TET and not those of ALLO and PHA. FIGS. 5 and 6present the evidence. As can be seen, there is an exact concordancebetween the T helper cell responses of PBL from these patients to FLU,TET and XENO. Thus, the T helper cell response to XENO (a non-recallantigen) can be substituted for the T helper reponses to FLU and TET(both of which require previous immunization).

EXAMPLE 2 Determining the Effect of an Agent on Immune System

Apart from its utility in determining subtle changes on immune system,the present invention is also useful for assessing the effect of anagent on the immune system (for example, an immunosuppressive drug). Todemonstrate this utility, the effect of cyclosporin A (CsA), one of themost extensively used immunosuppressive drugs for organ transplantationand treatment of autoimmune diseases, was studied. FIG. 7 shows theresults obtained when CsA was added to the in vitro culture. At thelowest concentrations of CsA (0.001 μg/ml), there was no detectableeffect on T helper cell function. At the intermediate concentrations ofCsA (0.003-0.01 μg/ml), there appeared to be a selective inhibition ofT4 helper activity, as demonstrated by the selective loss of responsesto FLU and TET, but not to ALLO. The highest concentrations of CsA used(0.03-0.1 μg/ml), abolished T helper activity to FLU and TET and reducedthe response to ALLO. These results indicate that different doses of CsAcan selectively affect distinct T helper cell pathways, and that thesensitive test system of the present invention can detect thesedifferences. It has also been found that treatment of kidney allograftpatients or patients with autoimmune uveitis with certain doses of CsAselectively abrogates T4 T helper cell function (data not shown).

Although the response to ALLO utilizes both the T4 and T8 helper cellpathways, the system of the present invention can be modified so thatone can use the ALLO response to detect T4 and T8 helper activitytogether, or to detect T4 helpers alone, or T8 helpers alone. Thismodification comprises depletion of the responding PBL of APC byadherence to plastic tissue culture flasks (Costar. Inc., Cambridge,Mass.) and nylon fiber (Fenwal Laboratories, Deerfield, Ill.), prior tostimulation with ALLO. Such treatment results in abrogation of theT4-self restricted T helper pathway, and the ALLO response that resultsis mediated by T8 helper cells. Alternatively, depletion of theallogeneic stimulator cells by adherence to plastic and nylon fiberresults in abrogation of the T8 helper response, and the resulting ALLOresponse is mediated by self-restricted T4 helper cells. The use of thisapproach is important because it permits analysis of distinctallo-specific T helper cell pathways, which are relevant for detectingpathway-selective effects of immuno-suppressive drugs, as well as fordetecting the T helper cell pathways that are responsible for foreigntissue graft rejection.

In summary, a sensitive in vitro test has been developed for T helpercell function using lymphokine production (IL-2) to the recall antigensinfluenza A virus, tetanus toxoid, and to alloantigens, and mousexenogeneic antigens. Using this system, one can detect a spectrum ofsubtle immune defects that occur in asymptomatic, HIV-infected patients,in patients with autoimmune diseases, in lymphoid cancers, as well as inkidney allograft patients who are on immuno-suppressive drugs.Furthermore, the sensitivity of this system lends it as a prognosticindicator for progression toward AIDS, and as a predictor of graftrejection. Based on the data presented herein, the value of the presentinvention to the medical community for providing a sensitive andaccurate evaluation of immune function (that is of minimal risk anddiscomfort to the patient) for several clinical conditions that involveimmune dysregulation is quite apparent.

Some specific applications of the principles and methodology describedherein are now illustrated.

Preparation of a Kit for Testing In Vitro T Helper-Antigen PresentingCell Function

In accordance with the present invention, a kit has been prepared thatcould be used for testing T helper cell function. Photographs of thiskit and a modification of it are shown in FIGS. 8 and 9. A 96-well,sterile, flat bottom, clear, plastic plate (with lid) of the typeproduced by Costar, Cambridge, Mass., catalog number 3596 is used. Theplate can be divided into eight rows, with four groups of triplicatewells in each row. To each of the eight rows would be added leukocytesfrom a single blood donor (patient or control donor) (at 1×10⁵ to 3×10⁵leukocytes per well in a 0.1 ml volume). These rows of leukocytes wouldbe testified for proliferative or IL-2 responses when unstimulated(medium, MED), or when stimulated with tetanus toxoid (TET), irradiatedforeign or "allogeneic" leukocytes (ALLO), or with the T cell mitogenphytohemagglutinin (PHA). The medium and various stimuli are added in0.1 ml volume, diluted in culture medium containing 5% pooled humanplasma from healthy donors. Thus, each row of 12 wells permitstriplicate cultures of leukocytes from one patient for one group ofunstimulated cells, and for groups of cells activated with the threedifferent stimuli (TET vs ALLO vs PHA). Each of the eight rows of 12wells permits the testing of eight different patients per plate. Thecultures are stimulated in a moist, 87° C., 3% CO₂ Incubator.

The cultures tested for IL-2 production should contain the anti-TAC IL-2receptor monoclonal antibody to block IL-2 production: the cultures tobe assayed for proliferation should not contain the anti-TAC reagent(Clerici, et al. 1989 J. Clin. Invest. 84:1892). Supernatants from theIL-2 cultures are collected, diluted and assayed on the IL-2-dependentCTLL line, for content of IL-21. Cells in the proliferative cultures arepulsed with ³ H-thymidine for 18 hours, harvested and the ³ H-thymidineis determined.

As illustrated in FIG. 8, each 96-well plate permits the testing ofT-helper cells' function for eight different patients for negativecontrol and for three different T-cell stimuli. Such analysis permitsfunctional T-helper cell characterization that detects four distinctcategories of immune function. The total number of PBL required perpatients for this analysis is in the range of 1.2×10⁶ to 3.6×10⁶. Todemonstrate clinical application, the system of the present inventionwas used to successfully characterize T-helper cell function in 30pediatric AIDS patients for whom the number of leukocytes is verylimiting (data not shown). Thus, the system has been scaled to a levelthat permits the testing of patients who can provide only small volumesof blood.

Of course, as mentioned above, the kit employs standardized preparationof various stimuli. Thus, frozen preparation of concentrated or dilutedpreparations of such stimuli as TET, ALLO and PHA are provided.Therefore, a modification of the kit would be to prepare 96-well platesin which optimal concentrations of lypholyzed TET and PHA are alreadycontained in the plates, and only PBL and medium would need to be added.However, it would not be possible to add lypholysed HLA allogeneic cellsto these plates because the particular T helper cell defect, which canbe detected using ALLO, requires intact and viable allogeneic antigenpresenting cells.

Another modification would be a kit in which alloantigenic stimulationis omitted from the test kit, which permits the use of lypholyzed TETand PHA (see FIG. 2). Clearly, this modification would exclude thedetection of one of the categories of immune dysfunction.

A protocol for the treatment of asymptomatic, HIV⁺ patients with AZT wasinitiated by the NIAID, NIH in 1988, and was terminated in August 1989,because it appeared that AZT therapy was effective in retarding theprogression of the asymptomatic patients toward symptomatic AIDS. Ourlaboratory was able to obtain blood samples before and one month afterinitiation of AZT treatment of a small number of asymptomatic patientsenrolled in this protocol. The limited and preliminary results shown inFIG. 3 indicate that after only one month of AZT therapy, in vitro testsfor T helper cell function indicated a significant restoration of immunefunction. Such results clearly indicate that the kit described hereincan be used to detect improved immune function resulting from drugtherapy, even as early as one month after initiation of therapy.

Early Detection of Kidney Allograft Rejection

In order to determine the reliable predictability of the system of thepresent invention, blood leukocytes from six patients who weretransplanted with foreign kidney grafts were tested. The findings,summarized in Table 4, indicate that only two of the six patients wereresponsive to HLA alloantigens (ALLO) by the CD4⁻ Th-self pathway. Incontrast, five of the six patients were responsive via their CD4⁻Th-alloAPC pathway, and all six were responsive via the CS8⁻ Th-alloAPCpathway. Most remarkable was the fact that two of the patients wereundergoing chronic kidney graft rejection, and these two patients werethe only ones (#2 and #3) whose leukocytes responded to HLA alloantigensvia the CD4⁻ Th-selfAPC pathway. There was no correlation betweenrejection, and the presence or absence of either of the other twopathways. It should be noted that the in vitro Th-APC tests wereperformed by a scientist who did not know that two patients wereundergoing chronic graft rejection. These results indicate that this invitro Th-APC pathway test is predictive for foreign tissue graftrejection in patients. The fact that the test was a predictor ofrejection even in chronic rejectors and did not require an acuterejection phase, also indicates that this test is very sensitive and mayserve as an early warning for graft rejection.

A method for predicting organ transplant rejection in accordance withthe present invention, comprises the steps of: (a) obtaining a sample ofperipheral blood leukocytes from an organ transplant recipientcandidate; (b) then determining in said sample the presence of immunedeficiency in the 4- self pathway by stimulating said leukocytes withalloantigen after depleting responder leukocytes of antigen-presentingcells, the presence of immune deficiency in said 4- self pathway beingpredictive of non-rejection of organ transplant and the absence ofimmune deficiency in said 4- self pathway being predictive of acute orchronic organ transplant rejection.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

                  TABLE 1    ______________________________________    Lack of Correlation Between T.sub.H Function    and CD4.sup.+  Cell Number           RANGE OF CD4.sup.+  CELL NUMBERS (/mm.sup.3):    Functional                              STATIS-    category*             400-600  600-800  >800  TOTAL  TICS    ______________________________________    By IL-2    production:    +/+/+    6        2        4     12     x.sup.2 = 4.60    -/+/+    14       16       10    40     p > 0.50    -/-/+    4        5        3     12    -/-/-    7        2        1     10    Total:   31       25       18    74    By    proliferation:    +/+/+    3        2        5     10     x.sup.2 = 7.73    -/+/+    11       13       4     28     p > 0.25    -/-/+    3        4        2      9    -/-/-    2        0        0      2    Total:   19       19       11    49    ______________________________________     *+/+/+ indicates patients who responded to FLU, TET, ALLO, and PHA;     -/+/+ indicates patients who responded to ALLO and PHA, but not to FLU an     TET;     -/-/+ indicates patients who responded to PHA, but not to FLU, TET, or     ALLO;     -/-/- indicates patients who did not respond to any of the four stimuli.

                  TABLE 2    ______________________________________    Lack of Correlation between T.sub.H    Function and Walter Reed Stage (WR)    Functional  WR    category*   1       2      TOTAL   STATISTICS    ______________________________________    By IL-2 production:    +/+/+       6       6      12      .sup.2 = 1.71    -/+/+       22      18     40      p > 0.50    -/-/+       6       6      12    -/-/-       8       2      10    Total:      42      32     74    By proliferation:    +/+/+       3       7      10      .sup.2 = 3.84    -/+/+       15      13     28      p > 0.25    -/-/+       5       4      9    -/-/-       2       0      2    Total:      25      24     49    ______________________________________     *+/+/+ indicates patients who responded to FLU, TET, ALLO, and PHA;     -/+/+ indicates patients who responded to ALLO and PHA, but not to FLU an     TET;     -/-/+ indicates patients who responded to PHA, but not to FLU, TET, or     ALLO;     -/-/- indicates patients who did not respond to any of the four stimuli.

                  TABLE 3    ______________________________________    Evidence for a Progressive and Sequential Loss of T.sub.H    Function in More Advanced Walter Reed Stages (WR)*                 Fraction and percentage                 of patients in:    T.sub.H response to:                       WR         WR    FLU    ALLO        1 and 2    3-6    ______________________________________    +      +           12/74 (16%)                                  0/22 (0%)    -      +           40/74 (54%)                                  8/22 (36%)    -      -           22/74 (30%)                                  14/22 (64%)    ______________________________________     *x.sup.2 = 9.82; p < 0.07

                  TABLE 4    ______________________________________    Correlation between a positive response by the    CD4.sup.+  Th-self APC pathway and chronic kidney graft rejection.    Th-APC pathway response    Patient           CD4.sup.+ Th-                     CD4.sup.+ Th-                               CD8.sup.+ Th-                                       Rejection of    Number selfAPC   alloAPC   alloAPC Kidney?    ______________________________________    1      negative  positive  positive                                       No    2      positive  positive  positive                                       Yes    3      negative  positive  positive                                       No    4      negative  positive  positive                                       No    5      positive  positive  positive                                       Yes    6      negative  negative  positive                                       No    ______________________________________

What is claimed is:
 1. A method for predicting organ transplantrejection, said method comprising:obtaining a sample of peripheral bloodleukocytes from an organ transplant recipient; exposing said peripheralblood leukocytes to allogeneic peripheral blood leukocytes expressingHLA alloantigens, said allogeneic peripheral blood leukocytes havingbeen depleted of antigen-presenting cells; and measuring the productionof IL-2 by the CD4⁺ T helper cells of said peripheral blood leukocytesin response to said allogeneic peripheral blood leukocytes;wherein:non-responsiveness by the CD4⁺ T helper cells of said peripheral bloodleukocytes being predictive of non-rejection of organ transplant; andresponsiveness by the CD4⁺ T helper cells of said peripheral bloodleukocytes being predictive of acute or chronic organ transplantrejection.